Abstract

Regulated intramembrane proteolysis of the amyloid precursor‐protein (APP) produces both a characterstic amyloid‐β peptide that contributes to neuritic plaque formation and neurodegeneration in Alzheimer disease and a small APP intracellular domain (AICD) that transcriptionally activates genes implicated in Alzheimer disease pathology. Although the biochemical events leading to amyloidogenic APP processing at the cell membrane have been described in detail, comparably little is known about the mechanistic basis of AICD‐dependent gene regulation in the nucleus. In this study, we show that the AICD activates transcription by targeting MED12, an RNA polymerase II transcriptional Mediator subunit that is implicated in human cognitive development. The AICD binds to MED12/Mediator in vitro and in vivo. Disruption of the AICD/MED12 interaction inhibits AICD transactivation potential and expression of AICD target genes. Mediator, in a MED12‐dependent manner, occupies only AICD‐bound promoter DNA, indicating that the AICD recruits Mediator to activate transcription. These results identify the MED12 interface in Mediator as a crucial transducer of AICD transactivation and a potential therapeutic target in Alzheimer disease.

Introduction

The amyloid precursor protein (APP) and two APP‐like proteins—APLP1 and APLP2—comprise a family of type I integral membrane proteins with essential and overlapping roles in brain development and function (Zheng & Koo, 2006; Walsh et al, 2007). Among its family members, APP is notable for its association with Alzheimer disease, a progressive neurodegenerative disorder characterized by early short‐term memory deficits and eventual loss of cognitive and executive functions. The pathological characteristics of Alzheimer disease are the presence of extracellular neuritic plaques enriched in amyloid‐β peptides and intracellular neurofibrillary tangles comprising hyperphosphorylated tau protein (Mucke, 2009). Amyloid‐β production through amyloidogenic processing of APP by membrane‐bound β‐ and γ‐secretases results in the simultaneous production of a 50‐amino‐acid carboxy‐terminal fragment corresponding to the APP intracellular domain (AICD; Haass, 2004). APLP1 and APLP2 are proteolytically processed in a mechanistically similar way, although sequence divergence within their amyloid‐β regions precludes plaque formation (Scheinfeld et al, 2002; Li & Sudhof, 2004). Notably, the AICD is among the most highly conserved protein regions found in APP family members, suggesting that it is functionally important.

The AICD has been reported to translocate into the nucleus and activate gene transcription (Cao & Sudhof, 2001; Gao & Pimplikar, 2001). Notably, several AICD‐target genes have been implicated in cellular processes relevant to Alzheimer disease. For example, neprilysin—a synaptic ectoenzyme that degrades amyloid‐β in the extracellular space—helps to maintain the concentration of amyloid‐β below the threshold for self‐aggregation, whereas p53‐mediated apoptosis has been implicated in Alzheimer‐disease‐associated neuronal death (Pardossi‐Piquard et al, 2005; Alves da Costa et al, 2006). More recently, a cohort of AICD‐target genes implicated in control of cytoskeletal dynamics, including α2‐actin, IGFBP3 and TAGLN, was found to be upregulated in the frontal cortex of Alzheimer disease patients, whereas another AICD‐regulated gene, Aquaporin 1, was shown to be upregulated in astrocytes surrounding amyloid‐β plaques within the brains of Alzheimer disease patients (Muller et al, 2007; Huysseune et al, 2009). Together, these observations suggest that AICD‐dependent nuclear signalling might contribute to the pathophysiology of Alzheimer disease.

Little is known about the mechanism by which the AICD activates gene transcription in the nucleus. Established interactions between the AICD, the transcriptional adaptor Fe65 and acetyltransferase Tip60 suggest it is linked to histone modification (Cao & Sudhof, 2001), which is supported by work documenting AICD‐dependent histone acetylation on the neprilysin promoter (Belyaev et al, 2009). However, a physical or functional AICD target within the RNA polymerase II general transcription machinery suggesting a direct mechanistic basis for gene regulation has not been reported so far. In this paper, we identify the MED12 subunit within the RNA polymerase II transcriptional Mediator as a direct physical target and functional transducer of the AICD, as well as the intracellular domains of APLP1 and APLP2. Mediator is a conserved multi‐subunit signal processor through which regulatory information conveyed by gene‐specific transcription factors is transduced to RNA polymerase II in order to alter gene expression programmes controlling cell growth, homeostasis, development and differentiation (Kornberg, 2005; Malik & Roeder, 2005). MED12 is implicated in vertebrate neuronal development, and genetic variation in MED12 is associated with cognitive and behavioural dysfunction in humans (Philibert et al, 2001; Wang et al, 2006; Risheg et al, 2007; Schwartz et al, 2007). Our findings that MED12 mediates the AICD transactivation potential and expression of AICD‐target genes identify MED12 as a new component in the APP‐dependent nuclear‐signalling pathway, and further implicate Mediator in a range of developmental and pathological processes driven by APP signal transduction.

Results

APP/APLP ICDs bind to MED12/Mediator

To clarify the role of MED12 in neural‐specific transcription control, we processed the MED12 C‐terminus (MED12C; amino acids 1,616–2,177) through a yeast two‐hybrid screen using a human foetal brain cDNA library (Zhou et al, 2006) and recovered APLP1, a neural‐restricted APP‐family member (Lorent et al, 1995). To confirm a physical interaction between MED12 and APLP1 and map their reciprocal binding domains, we tested the ability of glutathione S‐transferase (GST)–MED12 and GST–APLP1 derivatives to bind to a panel of APLP1 and MED12 truncation fragments, respectively. This analysis identified the principal reciprocal interaction surfaces on each protein which include a proline‐, glutamine‐ and leucine‐rich (PQL) domain on MED12 (amino acids 1,616–2,050) and the C‐terminal 50 amino acids of APLP1 corresponding to its transcriptionally active intracellular domain (ICD50; Fig 1A; supplementary Figs S1 and S2 online). As the ICDs of all three APP family members are highly conserved, we tested and confirmed that MED12 could also bind to APP and APLP2 ICD50 (Fig 1A; supplementary Figs S1 and S2 online). Thus, MED12 binds specifically through its PQL domain to the transcriptionally active ICDs of all three APP/APLP family members.

To determine whether APP/APLP ICDs interact with intact Mediator, we immunoprecipitated Mediator from nuclear extracts of HeLa cells transiently expressing APP/APLP ICDs and examined the immunoprecipitates for the presence of each ICD by western blot assay. This analysis revealed specific coimmunoprecipitation of each APP/APLP ICD50 with intact Mediator, confirming their association in mammalian cells (Fig 1B–D). Notably, Fe65 and Tip60—established co‐activators of the AICD—were coprecipitated with Mediator in the presence, but not the absence, of coexpressed AICD, demonstrating that Fe65 and Tip60 can form an AICD‐dependent multimeric complex with Mediator in mammalian cells (Fig 1E,F).

APP/APLP ICDs functionally interact with MED12

If the APP/APLP ICDs functionally target the MED12 interface in Mediator, their corresponding binding (PQL) domain on MED12 might inhibit their respective transactivation potentials through dominant‐negative interference. To test this possibility, we examined the influence of MED12–PQL expression on the transactivation activity of each APP/APLP ICD tethered to the GAL4 DNA‐binding domain (Cao & Sudhof, 2001). Ectopic expression of the MED12–PQL domain, but not of its neighbouring MED12–OPA domain, to which APP/APLP ICDs do not bind (supplementary Fig S2 online; data not shown), efficiently inhibited the transcriptional activities of all three APP family ICDs (Fig 2A).

To further examine the functional interaction between APP/APLP ICDs and MED12/Mediator, we monitored the influence of RNA interference‐mediated MED12 suppression on the transactivation activity of the ICD of each APP family member. Depletion of MED12 strongly inhibited the transcriptional activity of each APP/APLP ICD (Fig 2B,D). By contrast, depletion of MED12 had no influence on the transactivational activity of the herpes simplex virus VP16 transactivation domain (Fig 2B), the established targets of which in Mediator are MED17/25 (Ito et al, 1999). Thus, the influence of MED12 knockdown in this context seems to be restricted to the APP‐family ICDs. To confirm that MED12 mediates transactivation by the AICD through direct interaction, we assessed the respective abilities of short‐interfering RNA (siRNA)‐resistant wild‐type MED12 (MED12r) and a siRNA‐resistant AICD–binding‐defective MED12 derivative lacking the PQL domain (MED12r–ΔPQL) to rescue the AICD transactivation in MED12‐depleted cells. When expressed at equivalent levels, MED12r, but not MED12r–ΔPQL, elicited efficient rescue of AICD‐directed transactivation (Fig 2C,D). This finding precludes the possibility that reduced AICD‐driven transactivation elicited by MED12 siRNA derives from a nonspecific ‘off‐target’ effect, and confirms that the AICD physically interacts with the MED12 interface in Mediator to activate transcription.

MED12 mediates AICD‐dependent gene expression

To examine the role of MED12/Mediator as a transducer of AICD‐dependent nuclear signalling in a more physiological context, we monitored the effect of MED12 depletion on the expression levels of AICD‐regulated genes in three different human neuroblastoma cell lines: SK–N–SH (Tachida et al, 2008), SK–N–AS (Tillement et al, 2006) and NB7 (Belyaev et al, 2009). Of those genes identified as targets of AICD‐dependent regulation from previous functional studies (Pardossi‐Piquard et al, 2005; Alves da Costa et al, 2006; Muller et al, 2007), we confirmed four as targets of presenilin‐ and AICD‐dependent regulation in neuroblastoma cells (Neprilysin (NEP), Aquaporin 1, Microtubule‐associated monoxygenase, calponin and LIM domain containing 2 (MICAL2) and Fibronectin 1). Thus, pharmacological inhibition of γ‐secretase with DAPT (Fig 3A–C) or RNAi‐mediated APP depletion (Fig 3D–I) significantly reduced the expression levels of NEP, Aquaporin 1, MICAL2 and Fibronectin 1 in at least two out of the three neuroblastoma cell lines examined. Notably, RNAi‐mediated MED12 depletion also significantly reduced expression of these four genes in a manner perfectly concordant with their regulation by the AICD, implicating MED12/Mediator in AICD‐dependent gene regulation (Fig 3D–I). Finally, of these four genes only MICAL2 was coordinately regulated similarly in HeLa cells (data not shown), suggesting possible neural‐cell‐type‐specific gene regulation by the AICD and MED12/Mediator.

To explore the mechanistic relationship between the AICD and MED12/Mediator, we used chromatin immunoprecipitation (ChIP) to monitor their interdependence for occupancy of the NEP promoter. Depletion of AICD levels in NB7 cells through DAPT inhibition of γ‐secretase activity significantly reduced NEP promoter occupancy by the AICD, as expected, as well as by MED12/Mediator (Fig 4A–C). By contrast, RNAi‐mediated MED12 depletion reduced occupancy of the NEP promoter by Mediator, but not by the AICD, suggesting that the AICD recruits Mediator to the NEP promoter in a MED12‐dependent manner (Fig 4D–F).

Discussion

Despite increasing evidence for support a nuclear function for the AICD in gene regulation, none of the more than 20 previously reported AICD interaction partners represent a direct link to the RNA polymerase II general transcription apparatus. It has thus been difficult to determine how the AICD might activate transcription. Our identification of MED12/Mediator as a direct physical and functional target of the AICD suggests a simple solution to this problem. We propose a model in which promoter‐bound AICD recruits Mediator through its MED12 interface to stimulate the assembly, activation and/or regeneration of transcription complexes on its target genes (Malik & Roeder, 2005). Alternatively, or additionally, Mediator might facilitate the imposition of an AICD‐driven transcriptionally permissive chromatin environment (Ding et al, 2008).

Similarly to APP, MED12 is expressed in both developing and adult vertebrate tissues (Rocha et al, 2010). Nevertheless, early developmental phenotypes of MED12 knockout mice support a gene‐specific, as opposed to a ubiquitous, function for MED12 in RNA polymerase II transcription. For example, although MED12‐deficient mouse embryos die before completing gastrulation, they nevertheless develop further than those lacking the core Mediator subunits required for RNA polymerase II activity (Tudor et al, 1999; Rocha et al, 2010). Furthermore, MED12 hypomorphic mutant embryos support unperturbed development of many embryonic structures, processes and marker genes (Rocha et al, 2010). The broad distribution of MED12 might therefore endow Mediator with a unique interface through which it might be selectively recruited by diverse gene‐specific transcription factors, including the AICD.

Notably, among the 33 subunits that comprise Mediator, MED12 is uniquely implicated in vertebrate neural development and disease. In zebrafish, MED12 has been shown to be required for proper development of the brain and neural crest, among other organs, where it is important in the production of monoaminergic neurons and cranial sensory ganglia (Wang et al, 2006). In humans, polymorphisms in the Xq13 gene encoding MED12 have been linked with neuropsychiatric illnesses including schizophrenia and pyschosis, and MED12 mutations have been shown to cause two X‐linked mental retardation disorders: FG syndrome and Lujan syndrome (Philibert et al, 2001; Risheg et al, 2007; Schwartz et al, 2007). Our identification of MED12 as a direct ingress in Mediator for AICD‐dependent nuclear signalling suggests a possible link between the biological function of APP in brain development and disease and its role in transcriptional regulation. Finally, because genetic variation in MED12 is causally linked with cognitive and behavioural dysfunction in humans, it will be interesting to determine whether polymorphisms in MED12 might also predispose certain individuals to Alzheimer disease.

Quantitative real‐time reverse transcription–PCR. RNA extracted from cells 48 h after addition of DAPT (Calbiochem/EMD Biosciences) or 72 h after siRNA transfections was reverse‐transcribed using oligo (dT) and superscript iii (invitrogen). Quantitative PCR was performed using a 7900HT fast real‐time PCR system (Applied Biosystems) and Absolute SYBR Green ROX mix (ABgene). For each sample, the amplification plot and the corresponding dissociation curves were examined. Primer sequences are available on request.

Immunoprecipitations and ChIP. HeLa cells were collected 48 h after transfection with or without pCS3+H6T7‐ICDs (APP/APLP), pM‐Fe65 and pM‐Tip60. Nuclear extracts—prepared as described previously (Ding et al, 2008)—were adjusted using 200 mM KCl and 0.2% NP‐40, followed by incubation for 12 h at 4°C with 3 μg of either MED4‐specific rabbit polyclonal antibody or rabbit IgG. Immune complexes recovered by incubation with protein A‐sepharose (20 μl) for 2 h at 4°C were washed five times for 5 min each with 0.3‐M KCl D buffer (0.2% NP‐40), eluted in Laemmli sample buffer and resolved on 4–20% tris–glycine gradient gels (Lonza) for immunoblot analysis. Chromatin immunoprecipitation was performed as described previously (Ding et al, 2008) from NB7 cells treated with 0 or 25‐μM DAPT or control, APP, or MED12‐specific siRNAs using 3 μg of control or specific antibodies. Purified DNA was analysed by conventional or real‐time PCR. Primer sequences have been described previously (Belyaev et al, 2009).